368 Hydrophobia, Lyssa, or Rabies 



Williams and Lowden* stained Negri bodies by one of the follow- 

 ing methods: 



(a) Giemsa's solution. — The smears are fixed in methyl alcohol for about 5 

 minutes. The staining solution recommended is that last used by Giemsa : 



Azur II. Eosin 3.0 



Azur II 0.8 



Glycerin (Merck's chemically pure) 250.0 



Methyl alcohol (chemically pure) 250.0 



Both the glycerin and methyl alcohol are heated to 6o°C. The dyes are 

 put into the alcohol and the glycerin is added slowly, stirring. The mixture is 

 allowed to stand at even temperature over night, and after filtration is ready 

 for use. At the time of use one drop of the stain is added for every cubic centi- 

 meter of distilled water made alkaline by the addition of one drop of a i per 

 cent, solution of potassium carbonate to 10 cc. of the water. 



The stain is poured on the slide and allowed to stand for from one-half to three 

 hours. The longer time brings out the structure better and in twenty-four hours 

 well-made smears are not overstained. After the stain is poured off, the smear 

 is washed in running tap water for from one to three minutes and dried with 

 filter-paper. 



By this method the "bodies" are stained blue and the central bodies and 

 chromatoid granules blue, red or azure. The cytoplasm of the nerve cells stains 

 blue also, but the bodies can be seen distinctly within it. For diagnostic purposes 

 the method may be shortened thus: 



Methyl alcohol 5 minutes. 



Equal parts of Giemsa solution and distilled water 10 minutes. 



(6) The eosin-methylene blue of Mallory (q.v.). 



The smears are fixed in Zenkers' solution for one-half hour; after being rinsed 

 in tap water they are placed successively in 95 per cent, alcohol and iodine 

 for one-quarter hour, 95 per cent, alcohol for one-half hour, absolute alcohol 

 one-half hour, eosin solution 20 minutes, rinsed in tap water, methylene blue 

 solution 15 minutes; differentiated in 95 per cent, alcohol, lasting one to five 

 minutes and dried with filter-paper. 



With this method the cytoplasm of the "bodies" is magenta, light in the small 

 bodies, darker in the larger; the center bodies and chromatoid granules are a 

 very dark blue, the nerve-cell cytoplasm a light blue, the nucleus a darker blue 

 and the red blood-cells a brilliant eosin pink. 



Harris t uses the following method of staining Negri bodies that 

 seems to have the advantages of coloring them so as to bring out their 

 structure, and to do away with the granular precipitate that occurs 

 in most other methods. 



Smears of the appropriate material are made upon slides and fixed by the 

 application of methyl alcohol for one minute, are then washed with water to 

 remove the alcohol, placed for from one to three minutes in an old saturated 

 solution of eosin in 96 per cent, alcohol, after which they are washed for two or 

 three seconds with water to remove the excess of eosin. This stains the Negri 

 bodies. Counterstaining is effected by immersing for five to fifteen seconds 

 in a fresh solution of Unna's alkaline methylene blue, after which there is a brief 

 washing in water, decolorization in 95 per cent, alcohol and then the usual treat- 

 ment with absolute alcohol, xylol and balsam if the preparation is to be covered 

 and preserved, or the spread is blotted and dried if to be examined without a 

 cover. The whole process requires less than five minutes. 



Smears that have been dried for several days or weeks cannot be thus stained 

 with satisfaction. The older the eosin solution the more rapidly and intensely 

 it stains. To secure the best results it should not be less than two months old. 

 The methylene blue should not be more than a week or two old, else it will yield 

 an objectionable precipitate. 



*" Jour, of Infectious Diseases," 1906, in, 452. 

 t "Jour, of Infectious Diseases," 1908, v, 566. 



